Remedies for myeloma to be used together with nitrogen mustard antitumor agents

ABSTRACT

A therapeutic agent for myeloma comprising a combined use of a nitrogen mustard anticancer agent and anti-IL-6 receptor antibody. Thus, a therapeutic agent for myeloma comprising anti-IL-6 receptor antibody for use in combination with a nitrogen mustard anticancer agent; a therapeutic agent for myeloma comprising a nitrogen mustard anticancer agent for use in combination with anti-IL-6 receptor antibody; and a therapeutic agent for myeloma comprising a nitrogen mustard anticancer agent and anti-IL-6 receptor antibody.

TECHNICAL FIELD

The present invention relates to a pharmaceutical composition forcombined use of nitrogen mustard anticancer agents with anti-IL-6receptor antibody for treatment of myeloma.

BACKGROUND ART

For chemotherapy of human tumors, alkylating agents, antimetabolites,antitumor antibiotics, platinum compounds and the like have been used.When single uses of these activating agents do not exhibit markedtherapeutic effects, therapies in which multiple drugs are used incombination have been considered (Frei, E. III, Cancer Res. (1992) 32,2593-2607). As anticancer agents that belong to the alkylating agents,there are mentioned nitrogen mustard anticancer agents, which is ageneral term used for the anticancer agents that have a partialstructure called nitrogen mustard. Of the nitrogen mustard anticanceragents melphalan has been put into practical use.

IL-6 is a multifunctional cytokeine called B-cell stimulatory factor 2or interferon β2. IL-6 was discovered as a differentiation factorresponsible for activation of B-lymphocytes (Hirano, T. et al., Nature(1986) 324, 73-76). Thereafter, it was found to be a multifunctionalcytokeine that influences the function of various cells (Akira, S. etal., Adv. in Immunology (1993) 54, 1-78). IL-6 imparts its biologicalactivity through two proteins on the cell membrane.

One of them is a ligand-biding protein with a molecular weight of about80 kD, IL-6 receptor, to which IL-6 binds. IL-6 receptor occurs not onlyin a membrane-bound form that penetrates and is expressed on the cellmembrane but also as a soluble IL-6 receptor consisting mainly of theextracellular region. The other is non-ligand-binding gp130 with amolecular weight of about 130 kD that takes part in signal transmission.IL-6 and IL-6 receptor form a IL-6/IL-6 receptor complex, to whichanother membrane protein gp130 is bound, and thereby the biologicalactivity of IL-6 is transmitted to the cell (Taga et al., J. Exp. Med.(1987) 166, 967).

Antibodies to IL-6 receptor (anti-IL-6 receptor antibodies) have beenknown (Novick D. et al., Hybridoma (1991) 10, 137-146 Huang, Y. W. etal., Hybridoma (1993) 12, 621-630 International Patent ApplicationWO95-09873, French Patent Application FR 2694767, U.S. Pat. No.5,216,128), one of which is PM-1 derived from mice (Hirata et al., J.Immunology (1989) 143, 2900-2906). Furthermore, a reshaped antibodyobtained by replacing the complementarity determining regions (CDRs) ofthe mouse antibody with the CDRs of a human antibody has also beenknown.

However, the combined use of a nitrogen mustard anticancer agent andIL-6 receptor as a therapeutic agent for treatment of myeloma has notbeen known.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide a new type ofmyeloma therapeutic agent that is more effective than the conventionallyknown myeloma therapeutic agents.

As a result of an intensive study to solve the above problems, theapplicants have found that the combination of a nitrogen mustardanticancer agent, a conventionally known anticancer agent, and anti-IL-6receptor antibody has a synergistic effect, i.e. it is more effectivethan the sole use of the nitrogen mustard anticancer agent or the soleuse of anti-IL-6 receptor antibody for treatment of myeloma, and havecompleted the present invention.

Thus, the present invention provides a therapeutic agent for treatmentof myeloma comprising anti-IL-6 receptor antibody for use in combinationwith a nitrogen mustard anticancer agent.

The present invention also provides a therapeutic agent for treatment ofmyeloma comprising anti-IL-6 receptor monoclonal antibody for use incombination with a nitrogen mustard anticancer agent.

The present invention also provides a therapeutic agent for treatment ofmyeloma comprising PM-1 antibody for use in combination with a nitrogenmustard anticancer agent.

The present invention also provides a therapeutic agent for treatment ofmyeloma comprising a reshaped PM-1 antibody for use in combination witha nitrogen mustard anticancer agent.

The present invention also provides a therapeutic agent for treatment ofmyeloma comprising anti-IL-6 receptor antibody for use in combinationwith mechlorethamine, nitrogen mustard N-oxide, melphalan, uramustin,ifosfamide, chlorambucil, or cyclophosphamide.

The present invention also provides a therapeutic agent for treatment ofmyeloma comprising a reshaped human PM-1 antibody for use in combinationwith melphalan.

The present invention also provides a therapeutic agent for treatment ofmyeloma comprising a nitrogen mustard anticancer agent for use incombination with anti-IL-6 receptor antibody.

The present invention also provides a therapeutic agent for treatment ofmyeloma comprising a nitrogen mustard anticancer agent for use incombination with anti-IL-6 receptor monoclonal antibody.

The present invention also provides a therapeutic agent for treatment ofmyeloma comprising a nitrogen mustard anticancer agent for use incombination with PM-1 antibody.

The present invention also provides a therapeutic agent for treatment ofmyeloma comprising a nitrogen mustard anticancer agent for use incombination with a reshaped human PM-1 antibody.

The present invention also provides a therapeutic agent for treatment ofmyeloma comprising mechlorethamine, nitrogen mustard N-oxide, melphalan,uramustin, ifosfamide, chlorambucil, or cyclophosphamide in combinationwith anti-IL-6 receptor antibody.

The present invention also provides a therapeutic agent for treatment ofmyeloma comprising melphalan for use in combination with a reshapedhuman PM-1 antibody.

The present invention also provides a therapeutic agent for treatment ofmyeloma comprising a nitrogen mustard anticancer agent and anti-IL-6receptor antibody.

The present invention also provides a therapeutic agent for treatment ofmyeloma comprising a nitrogen mustard anticancer agent and anti-IL-6receptor monoclonal antibody.

The present invention also provides a therapeutic agent for treatment ofmyeloma comprising a nitrogen mustard anticancer agent and PM-1antibody.

The present invention also provides a therapeutic agent for treatment ofmyeloma comprising a nitrogen mustard anticancer agent and a reshapedhuman PM-1 antibody.

The present invention also provides a therapeutic agent for treatment ofmyeloma comprising mechlorethamine, nitrogen mustard N-oxide, melphalan,uramustin, ifosfamide, chlorambucil, or cyclophosphamide and anti-IL-6receptor antibody.

The present invention provides a therapeutic agent for treatment ofmyeloma comprising melphalan and a reshaped human PM-1 antibody.

BRIEF EXPLANATION OF DRAWINGS

FIG. 1 is a graph showing the relationship of anti-human IL-6 receptorantibody concentration and melphalan concentration with the growth(incorporation of ³H-labeled thymidine) of a human myeloma cell line inthe presence of 0.1 ng/ml IL-6.

FIG. 2 is a graph showing the relationship of anti-human IL-6 receptorantibody concentration and melphalan concentration with the growth(incorporation of ³H-labeled thymidine) of a human myeloma cell line inthe presence of 1 ng/ml IL-6.

FIG. 3 is a graph showing the relationship of anti-human IL-6 receptorantibody concentration and adriamycin concentration with the growth(incorporation of ³H-labeled thymidine) of a human myeloma cell line inthe presence of 0.1 ng/ml IL-6.

FIG. 4 is a graph showing the relationship of anti-human IL-6 receptorantibody concentration and adriamycin concentration with the growth(incorporation of ³H-labeled thymidine) of a human myeloma cell line inthe presence of 1 ng/ml IL-6.

FIG. 5 is a graph showing the relationship of anti-human IL-6 receptorantibody concentration and vincristine concentration with the growth(incorporation of ³H-labeled thymidine) of a human myeloma cell line inthe presence of 0.1 ng/ml IL-6.

FIG. 6 is a graph showing the relationship of anti-human IL-6 receptorantibody concentration and vincristine concentration with the growth(incorporation of ³H-labeled thymidine) of a human myeloma cell line inthe presence of 1 ng/ml IL-6.

FIG. 7 is a graph showing the survival days of the mice implanted withhuman myeloma cells in a single-drug administration (1 mg/kg) ofanti-human IL-6 receptor antibody (hPM-1) and melphalan, or in acombined use thereof.

FIG. 8 is a graph showing the amount of M protein in the mice implantedwith human myeloma cells in a single-drug administration (1 mg/kg) ofanti-human IL-6 receptor antibody (hPM-1) and melphalan, or in acombined use thereof.

FIG. 9 is a graph showing the survival days of mice implanted with humanmyeloma cells in a single-drug administration (3 mg/kg) of anti-humanIL-6 receptor antibody (hPM-1) and melphalan, or in a combined usethereof, with a synergistic effect obtained by the combined use.

FIG. 10 is a graph showing the changes in body weight of mice implantedwith human myeloma cells in a single-drug administration of anti-humanIL-6 receptor antibody (hPM-1) and melphalan, or in a combined usethereof.

FIG. 11 is a graph showing the amount of M protein in the serum of miceimplanted with human myeloma cells at 30 days after the tumorimplantation in the anti-human IL-6 receptor antibody (hPM1) single-drugadministration group and in the melphalan single-drug administrationgroup.

FIG. 12 is a graph showing the amount of M protein in the serum of miceimplanted with human myeloma cells at 35 days after the tumorimplantation in the melphalan single-drug administration group and theanti-human IL-6 receptor antibody (hPM1)-combined administration group.

FIG. 13 is a graph showing the amount of M protein in the serum of miceimplanted with human myeloma cells at 42 days after the tumorimplantation in the melphalan single-drug administration group and theanti-human IL-6 receptor antibody (hPM1)-combined administration group.

FIG. 14 is a graph showing the survival period by a survival curve ofmice implanted with human myeloma cells in the melphalan single-drugadministration group and the anti-human IL-6 receptor antibody(hPM1)-combined administration group, indicating an enhanced effect bycombined use.

FIG. 15 is a graph showing the changes in body weight of mice implantedwith human myeloma cells in the anti-human IL-6 receptor antibodysingle-drug administration group.

FIG. 16 is a graph showing the changes in body weight of mice implantedwith human myeloma cells in the melphalan single-drug administrationgroup and the anti-human IL-6 receptor antibody (hPM1)-combinedadministration group.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

Nitrogen mustard anticancer agents for use in the present invention is ageneral term for the anticancer agents having a partial structure callednitrogen mustard having the structure:

examples of which agents include:

Mechlorethamine,

Nitrogen mustard N-oxide(methyl-bis(β-chloroethyl)amine N-oxidehydrochloride),

Melphalan(p-[bis(2-chloroethyl)amino-L-phenylalanine),

Chlorambucil(p-bis(2-chloroethyl)aminophenylbutyric acid),

Uramustin(5-bis(2-chloroethyl)aminouracil),

Ifosmide(N,N′-bis(2-chloroethyl)-N′, O-propylenephosphoric acid esterdiamide

Cyclophosphamide(N,N′-bis(β-chloroethyl)-N′, O-propylenephosphoric acidester diamide,

and the like.

In accordance with the present invention, these nitrogen mustardanticancer agents may be used as a single drug or in combination. Amongthem, melphalan is also called sarcolysine or L-phenylalanine mustardand has the following structure:

Mechlorethamine can be obtained by a known method, for example, a methoddescribed in Abrams et al., J. Soc. Chem. Ind. (London) (1949) 68, 280.

Nitrogen mustard N-oxide can be obtained by a known method, for example,a method described in Aiko et al., J. Pharm. Soc. Japan (1952) 72, 1297.

Melphalan can be obtained by a known method, for example, a methoddescribed in Bergel, F. et al., J. Chem. Soc. (1954) 2409.

Chlorambucil can be obtained by a known method, for example, a methoddescribed in Balazc, M. K. et al., J. Pharm. Sci. (1970) 59, 563.

Uramustin can be obtained by a known method, for example, a methoddescribed in Lyttle and Petering, J. Am. Chem. Soc. (1958) 80, 6459.

Ifosfamide can be obtained by a known method, for example, a methoddescribed in Arnold H. et al., (1973 to Asta) U.S. Pat. No. 3,732,340,or Brassfield, H J. A. et al., J. Am. Che. Soc. (1975) 97, 4143.

Cyclophosphamide can be obtained by a known method, for example, amethod described in Arnold H. et al., Angew. Chem. (1958) 70, 539.

1. Anti-IL-6 Receptor Antibody

Anti-IL-6 receptor antibodies for use in the present invention may be ofany origin, any kind (monoclonal or polyclonal), and any form, as longas they have a higher therapeutic effect for myeloma when administeredin combination with a nitrogen mustard anticancer agent than when ananti-IL-6 receptor antibody alone is administered or when a nitrogenmustard anticancer agent alone is administered.

Anti-IL-6 receptor antibodies for use in the present invention can beobtained as polyclonal or monoclonal antibodies using a known method. Asthe anti-IL-6 receptor antibodies for use in the present invention,monoclonal antibodies of, in particular, a mammalian origin, arepreferred. Monoclonal antibodies of a mammalian origin include thoseproduced by a hybridoma and recombinant antibody produced by a hostwhich has been transformed with an expression vector containinggenetically engineered antibody genes. Anti-IL-6 receptor antibodies foruse in the present invention, via binding to IL-6 receptor, block thebinding of IL-6 to IL-6 receptor, and thereby inhibit signaltransmission of IL-6, and therefore are antibodies which inhibit thebiological activity of IL-6.

Examples of such antibodies include PM-1 antibody (Hirata, et al., J.Immunology (1989) 143, 2900-2906), or AUK12-20 antibody, AUK64-7antibody or AUK146-15 antibody (International Patent Application WO92-19759), and the like. Of them, PM-1 antibody is most preferred.

Incidentally, the hybridoma cell line which produces PM-1 antibody hasbeen internationally deposited under the provisions of the BudapestTreaty as PM-1 on Jul. 10, 1990 with the National Institute ofBioscience and Human Technology, Agency of Industrial Science andTechnology, of 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan, asFERM BP-2998.

2. Antibody Produced by Hybridoma

Monoclonal antibodies can be obtained by constructing a hybridoma usingbasically a known procedure as described bellow. Thus, IL-6 receptor isused as an immunizing antigen and is immunized in the conventionalmethod of immunization. The immune cells thus obtained are fused withknown parent cells in the conventional cell fusion process, and thenscreened by the conventional screening method to screen monoclonalantibody-producing cells.

Specifically, monoclonal antibodies may be obtained in the followingmanner.

For example, IL-6 receptor used as the immunizing antigen for obtainingantibody is not limited to any animal species, but IL-6 receptor derivedfrom humans is particularly preferred. For human IL-6 receptor, IL-6receptor protein can be obtained using a gene sequence disclosed inEuropean Patent Application EP 325474. There are two kinds of IL-6receptor: IL-6 receptor expressed on the cell membrane, and IL-6receptor detached from the cell membrane (Soluble IL-6 Receptor;Yasukawa et al., J. Biochem. (1990) 108, 673-676).

Soluble IL-6 receptor is composed mainly of the extracellular region ofIL-6 receptor bound to the cell membrane, and soluble IL-6 receptor isdifferent from the membrane-bound IL-6 receptor in that the former lacksthe transmembrane region or both of the transmembrane region and theintracellular region. In accordance with the present invention, IL-6receptor used as the immunizing antigen may be either the membrane-boundor the soluble IL-6 receptor. Alternatively, it may be a mutant thereof.

After a gene encoding IL-6 receptor is inserted into a known expressionvector to transform an appropriate host cell, the desired IL-6 receptorprotein is purified from the host cell or a culture supernatant thereofusing a known method, and the IL-6 receptor protein thus purified may beused as the immunization antigen. Alternatively, cells that express IL-6receptor protein may be used as the immunization antigen.

Preferably mammals to be immunized with the immunization antigen areselected in consideration of their compatibility with the parent cellsfor use in cell fusion and they generally include, but are not limitedto, rodents, logomorphas, and primates.

As rodents, for example, mice, rats, hamsters, etc. are used. Aslogomorphas, for example, rabbits are used. As primates, for example,monkeys are used. As monkeys, catarrhines (Old-World monkeys) such ascynomolgi (crab-eating macaque), rhesus monkeys, sacred baboons,chimpanzees etc. are used.

Immunization of animals with an immunization antigen is carried outusing a known method. A general method, for example, involvesintraperitoneal or subcutaneous administration of an immunizationantigen to the mammal. Specifically, an immunization antigen which wasdiluted and suspended in an appropriate amount of phosphate bufferedsaline (PBS) or physiological saline etc. is mixed with an appropriateamount of Freund's complete adjuvant. After being emulsified, it ispreferably administered to a mammal for several times every 4 to 21days. Additionally a suitable carrier may be used at the time ofimmunization of the immunization antigen.

After the immunization and confirmation of the increase in the desiredantibody levels in the serum by a conventional method, immune cells aretaken out from the mammal and are subjected to cell fusion, in whichespecially preferred immune cells are the spleen cells.

The mammalian myeloma cells as the other parent cells which aresubjected to cell fusion with the above-mentioned immune cellspreferably include various known cell lines such as P3 (P3x63Ag8.653)(Kearney, J. F. et al., J. Immunol. (1979) 123, 1548-1550), P3x63Ag8U.U1(Yelton, D. E. et al., Current Topics in Microbiology and Immunology(1978) 81, 1-7), NS-1 (Kohler, G. and Milstein, C., Eur. J. Immunol.(1976) 6, 511-519), MPC-11 (Margulies, D. H. et al., Cell (1976) 8,405-415), SP2/0 (Shulman, M. et al., Nature (1978) 276, 269-270), FO (deSt. Groth, S. F. and Scheidegger, D., J. Immunol. Methods (1980) 35,1-21), S194 (Trowbridge, I. S., J. Exp. Med. (1978) 148, 313-323), R210(Galfre, G. et al., Nature (1979) 217, 131-133) and the like.

Cell fusion between the above immune cells and the myeloma cells may beessentially conducted in accordance with a known method such as isdescribed in Milstein et al. (Galfre, G. and Milstein, C., MethodsEnzymol. (1981) 73, 3-46) and the like.

More specifically, the above cell fusion is carried out in theconventional nutrient broth in the presence of, for example, a cellfusion accelerator. As the cell fusion accelerator, for example,polyethylene glycol (PEG), Sendai virus (HVJ) and the like may be used,and an adjuvant such as dimethyl sulfoxide etc. may be added as desiredto enhance efficiency of the fusion.

The preferred ratio of the immune cells and the myeloma cells for useis, for example, 1 to 10 times more immune cells than the myeloma cells.Examples of culture media to be used for the above cell fusion includeRPMI 1640 medium and MEM culture medium suitable for the growth of theabove myeloma cell lines, and the conventional culture medium used forthis type of cell culture, and besides a serum supplement such as fetalcalf serum (FCS) may be added.

In cell fusion, predetermined amounts of the above immune cells and themyeloma cells are mixed well in the above culture liquid, to which a PEGsolution previously heated to about 37° C., for example a PEG solutionwith a mean molecular weight of 1000 to 6000, is added at aconcentration of 30 to 60% (w/v) and mixed to obtain the desired fusioncells (hybridomas). And then by repeating a sequential addition of asuitable culture liquid and centrifugation to remove the supernatant,cell fusion agents etc. that are undesirable for the growth of thehybridoma can be removed.

Said hybridoma is selected by culturing in the conventional selectionmedium, for example, HAT culture medium (a culture liquid containinghypoxanthine, aminopterin, and thymidine). Culturing in said HAT culturemedium is continued generally for the period of time sufficient toeffect killing of the cells other than the desired hybridoma (non-fusioncells), generally several days to several weeks. The conventionallimiting dilution method is conducted in which the hybridomas producingthe desired antibody are screened and monoclonally cloned.

In addition to obtaining the above hybridoma by immunizing an animalother than the human with an antigen, it is also possible to immunizehuman lymphocytes in vitro with IL-6 receptor protein or IL-6 receptorprotein-expressing cells, and the resulting immunized lymphocytes arefused with a myeloma cell, for example U266, having the ability ofdividing permanently to obtain a hybridoma that produces the desiredhuman antibody having the activity of binding to and neutralizing IL-6receptor (Japanese Post-examined Patent Publication (Kokoku) 1-59878).Furthermore, a transgenic animal having a repertoire of human antibodygenes is immunized with the antigen IL-6 receptor or IL-6receptor-expressing cells to obtain anti-IL-6 receptorantibody-producing cells. The cells are then fused with myeloma cells toobtain hybridomas that are used to obtain human antibody to IL-6receptor (see International Patent Application WO 92-03918, WO 93-12227,WO 94-02602, WO 94-25585, WO 96-33735 and WO 96-34096).

The monoclonal antibody-producing hybridomas thus constructed can bemaintained in the conventional culture liquid, or can be stored for aprolonged period of time in liquid nitrogen.

In order to obtain monoclonal antibodies from said hybridoma, there canbe mentioned a method in which said hybridoma is cultured in theconventional method and the antibodies are obtained as the supernatant,or a method in which the hybridoma is transplanted to and grown in amammal compatible with said hybridoma and the antibodies are obtained asthe ascites. The former method is suitable for obtaining highly purifiedantibodies, whereas the latter is suitable for a large scale productionof antibodies.

In addition to using a hybridoma for antibody production, immune cellssuch as antibody-producing immunized lymphocytes which has beenimmortalized with an oncogene can be used.

3. Recombinant Antibody

Monoclonal antibodies may be also obtained as a recombinant antibodywhich has been produced by the recombinant gene technology. For example,recombinant antibody can be obtained by cloning a gene of an antibodyfrom a hybridoma or an immune cell such as antibody-producing immunizedlymphocytes, and then integrated into a suitable vector, which is thenintroduced into a host to produce said antibody (see, for example,Borrebaeck, C. A. K. and Larrick, J. W., THERAPEUTIC MONOCLONALANTIBODIES, published in the United Kingdom by MACMILLAN PUBLISHERS LTD.1990).

Specifically, mRNA encoding the variable region (V region) of anti-IL-6receptor antibody is isolated from the hybridoma producing anti-IL-6receptor antibody. The isolation of mRNA is conducted by preparing totalRNA using, for example, a known method such as the guanidineultracentrifuge method (Chirgwin, J. M. et al., Biochemistry (1979) 18,5294-5299), the AGPC method (Chomczynski, P. and Sacchi, N., Anal.Biochem. (1987) 162, 156-159), and then mRNA is purified from the totalRNA using the mRNA Purification kit (Pharmacia) and the like.Alternatively, mRNA can be directly prepared using the Quick Prep mRNAPurification Kit (Pharmacia).

cDNA of the V region of antibody may be synthesized from the mRNA thusobtained using a reverse transcriptase. cDNA may be synthesized usingthe AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (SeikagakuKogyo), and the like. Alternatively, for the synthesis and amplificationof cDNA, the 5′-Ampli FINDER RACE Kit (Clontech) and the 5′-RACE method(Frohman, M. A. et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85,8998-9002; Belyavsky, A. et al., Nucleic Acids Res. (1989) 17,2919-2932) which employs polymerase chain reaction (PCR) may be used.

The desired DNA fragment is purified from the PCR product obtained andmay be ligated to vector DNA. Moreover, a recombinant vector isconstructed therefrom and then is transfected into E. coli etc., whichis selected to prepare the desired recombinant vector. The base sequenceof the desired recombinant vector may be confirmed by a known methodsuch as the dideoxy nucleotide chain termination method.

Once the DNA encoding the V region of the desired anti-IL-6 receptorantibody has been obtained, it may be ligated to DNA encoding theconstant region (C region) of the desired antibody, which is thenintegrated into an expression vector. Alternatively, DNA encoding the Vregion of the antibody may be integrated into an expression vector whichalready contains DNA encoding the C region of the antibody. The C regionof the antibody may be derived from the same animal species as that ofthe V region, or from a different animal species from that of the Vregion.

In order to produce anti-IL-6 receptor antibody for use in the presentinvention, the antibody gene is integrated into an expression vector soas to be expressed under the control of an expression regulatory region,for example an enhancer and/or a promoter. Subsequently, the expressionvector is transformed into a host cell and the antibody is thenexpressed therein.

The antibody gene may be expressed by integrating separately DNAencoding a heavy chain (H chain) and a light chain (L chain) of theantibody into an expression vector and co-transforming the host cell, orby integrating DNA encoding an H chain and an L chain into a singleexpression vector and transforming the host cell (International PatentApplication WO 94-11523).

4. Altered Antibody

As recombinant antibodies for use in the present invention, artificiallyaltered recombinant antibodies such as chimeric antibody and humanizedantibody can be used for the purpose of lowering xenogenic antigenicityagainst humans. Altered antibodies can have the C regions of humanantibody and antibodies such as chimeric antibody or humanized antibodycan be used. These altered antibodies can be produced using knownmethods.

Chimeric antibody can be obtained by ligating the thus obtained DNAencoding the V region of antibody other than human antibody to DNAencoding the C region of human antibody, which is then integrated intoan expression vector and introduced into a host for production of theantibody therein (see European Patent Application EP 125023, andInternational Patent Application WO 92-19759). Using this known method,chimeric antibody useful for the present invention can be obtained.

Plasmid coding for the V region of the L chain or the V region of the Hchain of PM-1 antibody has each been designated as pPM-k3 and pPM-h1,respectively, and E. coli having a respective plasmid has beeninternationally deposited under the provisions of the Budapest Treaty asNCIMB40366 and NCIMB40362 on Feb. 11, 1991 with the National Collectionsof industrial and Marine Bacteria Limited.

Humanized antibody which is also called reshaped human antibody has beenmade by transplanting the complementarity determining regions (CDRs) ofan antibody of a mammal other than the human, for example mouseantibody, into the CDRs of human antibody. The general recombinant DNAtechnology for preparation of such antibodies is also known (seeEuropean Patent Application EP 125023 and International PatentApplication WO 92-19759).

Specifically, a DNA sequence which was designed to ligate the CDRs ofmouse antibody with the framework regions (FRs) of human antibody issynthesized from several divided oligonucleotides having sectionsoverlapping with one another at the ends thereof, and theoligonucleotides are then synthesized into one integrated DNA. The DNAthus obtained is ligated to a DNA encoding a C region of human antibodyand then is integrated into an expression vector, which is introducedinto a host for antibody production (see European Patent Application EP239400 and International Patent Application WO 92-19759).

For the FRs of human antibody being ligated to CDRs, the FR that makeCDR a favorable antigen-binding site is selected. When desired, Aminoacids in the FR of antibody V region may be substituted so that the CDRof humanized antibody may form an appropriate antigen binding site(Sato, K. et al., Cancer Res. (1993) 53, 851-856).

A preferred embodiment of humanized antibody for use in the presentinvention includes humanized PM-1 antibody (see International PatentApplication WO92-19759). In the humanized PM-1 antibody, CDRs of thePM-1 antibody derived from a mouse have been ligated to the FRs of thehuman antibody REI for the L chain, and the FRs of the human antibodyNEW, and part of the amino acid residues of the FR has been substitutedto obtain antigen-binding activity.

In order to produce anti-IL-6 receptor antibody for use in the presentinvention, the antibody gene is integrated into an expression vector soas to be expressed under the control of an expression regulatory region,for example an enhancer and/or a promoter. Subsequently, the expressionvector is transformed into a host cell and the antibody is thenexpressed therein.

The antibody gene may be expressed by integrating separately DNAsencoding a heavy chain (H chain) and a light chain (L chain) of anantibody into an expression vector and co-transforming the host cell, orby integrating a DNA encoding an H chain and an L chain into a singleexpression vector and transforming the host cell (International PatentApplication WO 94-11523).

Chimeric antibody consists of the V regions of antibody derived from amammal other than the human and the C regions derived from humanantibody, whereas humanized antibody consists of the CDRs of antibodyderived from a mammal other than the human and the FRs and the C regionsof antibody derived from human antibody. Accordingly, since the aminoacid sequences derived from a mammal other than the human are reduced toa minimum in the above antibodies, antigenicity thereof in the humanbody is reduced so that they are useful as the active ingredient of thetherapeutic agents of the present invention.

As the C region of human antibody, there can be used, for example, Cγ1,Cγ2, Cγ3, or Cγ4. The C region of a human antibody may also be modifiedin order to improve the stability of antibody or of the productionthereof.

5. Antibody Fragments and Modified Antibody

Antibodies for use in the present invention may be fragments of antibodyor modified versions thereof as long as they bind to IL-6 receptor andthereby inhibit the binding of IL-6 and IL-6 receptor to block signaltransmission and to inhibit the biological activity of IL-6. They areantibody fragments or modified antibodies which, when used incombination with a nitrogen mustard anticancer agent, have a highertherapeutic effect for myeloma than IL-6 receptor antibody alone or anitrogen mustard anticancer agent alone.

For example, as fragments of antibody, there may be mentioned Fab,F(ab′)2, Fv or single-chain Fv (scFv) in. which Fv's of H chain and Lchain were ligated via a suitable linker. Specifically antibodies aretreated with an enzyme, for example, papain or pepsin, to produceantibody fragments, or genes encoding these antibody fragments areconstructed, and then integrated into an expression vector, which isexpressed in a suitable host cell (see, for example, Co, M. S. et al.,J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, A. H.,Methods Enzymol. (1989) 178, 476-496; Plucktrun, A. and Skerra, A.,Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol.(1986) 121, 652-663; Rousseaux, J. et al., Methods Enzymol. (1986) 121,663-669; Bird, R. E. and Walker, B. W., Trends Biotechnol. (1991) 9,132-137).

scFv can be obtained by ligating a V region of an H chain and a V regionof an L chain of an antibody. In the scFv, the V region of H chain andthe V region of L chain are preferably ligated via a linker, preferablya peptide linker (Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A.(1988) 85, 5879-5883). The V region of H chain and the V region of Lchain in the scFv may be derived from any of the above-mentionedantibodies. As the peptide linker for ligating the V regions, anysingle-chain peptide comprising, for example, 12-19 amino acid residuesmay be used.

DNA encoding scFv can be obtained using a DNA encoding an H chain or a Vregion of an H chain of the above antibody and a DNA encoding an L chainor a V region of an L chain of the above antibody as the template byamplifying the portion of the DNA encoding the desired amino acidsequence among the above sequences by the PCR technique with the primerpair specifying the both ends thereof, and by further amplifying thecombination of DNA encoding the peptide linker portion and the primerpair which defines that both ends of said DNA be ligated to the H chainand the L chain, respectively.

Once DNAs encoding scFv are constructed, an expression vector containingthem and a host transformed with said expression vector can be obtainedby the conventional methods, and scFv can be obtained using theresultant host by the conventional methods.

Antibody fragments may be those antibody fragments part of whichsequence has undergone mutation, substitution, deletion, or insertion.These antibody fragments can be produced by obtaining the gene thereofin a similar manner to that mentioned above and by allowing it to beexpressed in a host. “Antibody” as used in the claim of the presentapplication encompasses these antibody fragments.

As modified antibodies anti-IL-6 receptor antibody associated withvarious molecules such as polyethylene glycol (PEG) can be used.“Antibody” as used in the claim of the present application encompassesthese modified antibodies. These modified antibodies can be obtained bychemically modifying the antibodies thus obtained. These methods havealready been established in the art.

6. Expression and Production of Recombinant Antibody, Altered Antibody,and Antibody Fragment

Antibody genes constructed as mentioned above may be expressed andobtained in a known manner. In the case of mammalian cells, expressionmay be accomplished using an expression vector containing a commonlyused useful promoter, an antibody gene to be expressed, and DNA in whichthe poly A signal has been operably linked at 3′ downstream thereof.Examples of the promoter/enhancer include human cytomegalovirusimmediate early promoter/enhancer.

Additionally, as the promoter/enhancer which can be used for expressionof antibody for use in the present invention, there can be used viralpromoters/enhancers such as retrovirus, polyoma virus, adenovirus, andsimian virus 40 (SV40), and promoters/enhancers derived from mammaliancells such as human elongation factor 1α (HEF1α).

For example, expression may be readily accomplished by the method ofMulligan, R. C. et al. (Nature (1979) 277, 108-114) when SV40promoter/enhancer is used, and by the method of Mizushima, S. et al.(Nucleic Acids Res. (1990) 18, 5322) when HEF1α promoter/enhancer isused.

In the case of E. coli, expression may be conducted by operably linkinga commonly used promoter, a signal sequence for antibody secretion, andan antibody gene to be expressed, followed by expression thereof. As thepromoter, for example, there can be mentioned lacz promoter and araBpromoter. The method of Ward, E. S. et al. (Nature (1989) 341, 544-546;FASEB J. (1992) 6, 2422-2427) may be used when lacz promoter is used,and the method of Better, M. et al. (Science (1988) 240, 1041-1043) maybe used when araB promoter is used.

As a signal sequence for antibody secretion, when produced in theperiplasm of E. coli, the pelB signal sequence (Lei, S. P. et al., J.Bacteriol. (1987) 169, 4379-4383) can be used. After separating theantibody produced in the periplasm, the structure of the antibody isappropriately refolded before use (see, for example, InternationalPatent Application WO 96-30394).

As the origin of replication, there can be used those derived from SV40,polyoma virus, adenovirus, bovine papilloma virus (BPV), and the like.Furthermore, for gene amplification in the host cell system, expressionvectors can include as selection markers the aminoglycoside transferase(APH) gene, the thymidine kinase (TK) gene, E. coli xanthineguaninephosphoribosyl transferase (Ecogpt) gene, the dihydrofolatereductase (dhfr) gene, and the like.

For the production of antibody for use in the present invention, anyproduction system can be used, and the production system of antibodypreparation comprises the in vitro or the in vivo production system.

As the in vitro production system, there can be mentioned a productionsystem which employs eukaryotic cells and the production system whichemploys prokaryotic cells.

When eukaryotic cells are used, there are the production systems whichemploy animal cells, plant cells, and fungal cells. Known animal cellsinclude (1) mammalian cells such as CHO cells, COS cells, myeloma cells,baby hamster kidney (BHK) cells, HeLa cells, and Vero cells, (2)amphibian cells such as Xenopus oocytes, or (3) insect cells such assf9, sf21, and Tn5. Known plant cells include, for example, thosederived from the Nicotiana family, more specifically cells derived fromNicotiana tabacum which is subjected to callus culture. Known fungalcells include (1) yeasts such as the Saccharomyces family, morespecifically Saccharomyces cereviceae, or (2) mold fungi such as thegenus Aspergillus, more specifically Asperaillus niger.

When prokaryotic cells are used, there are the production systems whichemploy bacterial cells. Known bacterial cells include Escherichia coli,and Bacillus subtilis.

By introducing via transformation the gene of the desired antibody intothese cells and culturing the transformed cells in vitro, the antibodycan be obtained. Culturing is conducted in the known methods. Forexample, as the culture liquid for mammalian cells, DMEM, MEM, RPMI1640,IMDM and the like can be used, and serum supplements such as fetal calfserum (FCS) may be used in combination. In addition, antibodies may beproduced in vivo by implanting cells into which the antibody gene hasbeen introduced into the peritoneal cavity of an animal, and the like.

As in vivo production systems, there can be mentioned those which employanimals and those which employ plants. When animals are used, there arethe production systems which employ mammals and insects.

As mammals, goats, pigs, sheep, mice, and cattle can be used (Glaster,V., SPECTRUM Biotechnology Applications, 1993).

When mammals are used, transgenic animals can be used. For example,antibody genes are inserted into the gene encoding protein which isinherently produced in the milk such as goat β casein to prepare fusiongenes. DNA fragments containing the fusion gene into which the antibodygene has been inserted are injected to a goat embryo, and the embryo isintroduced into a female goat. The desired antibody is obtained from themilk produced by the transgenic goat borne to the goat who received theembryo or offsprings thereof. In order to increase the amount of milkproduced containing the desired antibody produced by the transgenicgoat, hormones may be given to the transgenic goat as appropriate.(Ebert, K. M. et al., Bio/Technology (1994) 12, 699-702).

Also as insects, silkworms can be used. When silkworms are used,baculovirus into which the desired antibody gene has been inserted isinfected to the silkworm, and the desired antibody can be obtained fromthe body fluid of the silkworm (Maeda, S. et al., Nature (1985) 315,592-594).

Moreover, when plants are used, tabacco, for example, may be used. Whentabacco is used, the desired antibody gene is inserted into anexpression vector for plants, for example pMON 530, and then the vectoris introduced into a bacterium such as Agrobacterium tumefaciens. Thebacterium is then infected to tobacco such as Nicotiana tabacum toobtain the desired antibody from the leaves of the tobacco (Ma, J. K. etal., Eur. J. Immunol. (1994) 24, 131-138).

An antibody gene is introduced, as mentioned above, into these animalsor plants, and then the antibody is produced in such animals and plantsand is collected therefrom.

When antibody is produced in in vitro or in vivo production systems, asmentioned above, DNAs encoding an H chain and an L chain of an antibodyare separately integrated into expression vectors and the hosts aretransformed simultaneously, or a DNA encoding an H chain and an L chainof an antibody is integrated into a single expression vector and thehost is transformed therewith (International Patent Application WO94-11523).

7. Separation and Purification of Antibody

Antibodies expressed and produced as described above can be separatedfrom inside or outside of the host cell and then may be purified tohomogeneity. Separation and purification of antibody for use in thepresent invention may be accomplished by methods of separation andpurification conventionally used for proteins without any limitation.

For example, separation and purification of antibody may be accomplishedby combining, as appropriate, column chromatography such as affinitychromatography, filtration, ultracentrifugation, salting-out, dialysis

and the like (Antibodies: A Laboratory Manual, Ed Harlow and David Lane,Cold Spring Harbor-Laboratory, 1988).

As the column used for affinity chromatography, there can be mentionedProtein A column and Protein G column. Examples of the column employingProtein A column are Hyper D, POROS, Sepharose F. F; (Pharmacia) and thelike.

Chromatography other than affinity chromatography includes, for example,ion exchange chromatography, hydrophobic chromatography, gel-filtration,reverse-phase chromatography, absorption chromatography and the like(Strategies for Protein Purification and Characterization: A LaboratoryCourse Manual, Ed Daniel R. Marshak et al., Cold Spring HarborLaboratory Press, 1996). Furthermore, said chromatography may be carriedout using a liquid-phase chromatography such as HPLC, FPLC, and thelike.

8. Measurement of Antibody Concentration

The concentration of antibody obtained as above can be determined bymeasurement of absorbance or by the enzyme-linked immunosorbent assay(ELISA) and the like. Thus, when absorbance measurement is employed, theantibody obtained is appropriately diluted with PBS and then theabsorbance is measured at 280 nm, followed by calculation using theabsorption coefficient of, though different with species and subclasses,1.4 OD at 1 mg/ml in the case of human antibody.

When the ELISA method is used, measurement is conducted as follows.Thus, 100 μl of goat anti-human IgG antibody diluted to 1 μg/ml in 0.1 Mbicarbonate buffer, pH 9.6, is added to a 96-well plate (Nunc), and isincubated overnight at 4° C. to immobilize the antibody. After blocking,100 μl each of appropriately diluted antibody of the present inventionor samples containing the antibody, or 100 μl of human IgG of a knownconcentration as the concentration standard is added, and incubated atroom temperature for 1 hour.

After washing, 100 μl of 5000-fold diluted alkaline phosphatase-labeledanti-human IgG antibody is added, and incubated at room temperature for1 hour. After washing, the substrate solution is added and incubated,followed by measurement of absorbance at 405 nm using the MICROPLATEREADER Model 3550 (Bio-Rad) to calculate the concentration of thedesired antibody based on the absorbance of the concentration standardIgG.

For determination of antibody concentration, BIAcore (Pharmacia) can beused.

9. Confirmation of the Activity of Antibody

Evaluation of activity of anti-IL-6 receptor antibody of the presentinvention can be conducted using a commonly known method. IL-6 is addedto a plate in which IL-6 responsive cells such as HN60.BSF2 cells werecultured. Then, evaluation is made in the presence of anti-IL-6 receptorantibody, using the incorporation of ³H labeled thymidine by IL-6dependent cells as an index.

Alternatively, ¹²⁵I-labeled IL-6 and anti-IL-6 receptor antibody areadded to a plate in which IL-6 receptor-expressing cells such as U266have been cultured and then the amount of ¹²⁵I-labeled IL-6 that isbound to the IL-6-expressing cells is determined for evaluation(Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold SpringHarbor-Laboratory, 1988).

As methods for determining the antigen-binding activity of anti-IL-6receptor antibody for use in the present invention, there can be usedELISA, EIA (enzymeimmunoassay), RIA (radioimmunoassay), or thefluorescent antibody method.

When ELISA is employed, for example, IL-6 receptor is added to a 96-wellplate onto which antibody against IL-6 receptor has been immobilized,and then samples containing the desired anti-IL-6 receptor antibody, forexample a culture supernatant of anti-IL-6 receptor antibody-producingcells or purified antibody, are added thereto. Secondary antibody thatrecognizes the desired anti-IL-6 receptor antibody, labeled with anenzyme such as alkaline phosphatase is added, and the plate isincubated, washed, and then the enzyme substrate such as p-nitrophenylphosphate is added thereto. Then the absorbance is measured to evaluatethe antigen-binding activity. A soluble IL-6 receptor may be used as theIL-6 receptor.

As methods for measuring the inhibition activity of ligand receptorbinding of the anti-IL-6 receptor antibody for use in the presentinvention, the conventional Cell ELISA or the ligand receptor bindingassay can be used.

In the case of Cell ELISA, for example, cells expressing IL-6 receptorare cultured in a 96-well plate and then immobilized withparaformaldehyde etc. Alternatively, membrane fractions of cellsexpressing IL-6 receptor are prepared and a 96-well plate on which IL-6receptors have been immobilized is prepared. To this are added a samplecontaining the desired anti-IL-6 receptor antibody, for example aculture supernatant of anti-IL-6 receptor antibody-producing cells, andpurified antibody, and IL-6 labeled with a radioisotope such as ¹²⁵I,and then the plate is incubated, washed, and radioactivity is measuredto determine the amount of IL-6 bound to the IL-6 receptor and therebyto evaluate the inhibition activity of ligand receptor binding ofanti-IL-6 receptor antibody.

In the inhibition assay of IL-6 binding to IL-6 receptor on the cells,cells expressing IL-6 receptors are separated by means of centrifugationetc. and resuspended to prepare a cell suspension. A solution of IL-6labeled with a radioisotope such as ¹²⁵I, or a mixture of unlabeled IL-6and labeled IL-6, and a solution containing anti-IL-6 receptor antibodywhose concentration has been adjusted are added to the cell suspension.After incubating for a certain period of time, the cells are separated,and the radioactivity of the labeled IL-6 bound to the cell is measured.

For evaluation of activity of the above antibody, BIAcore (Pharmacia)can be used.

10. Method of Administration and Pharmaceutical Preparation

In accordance with the present invention, a nitrogen mustard anticanceragent and anti-IL-6 receptor antibody are used in combination. “Used incombination” as used herein refers to a case in which pharmaceuticalcompositions are administered at different times, a case in whichpharmaceutical compositions are administered at the same time, and acase in which one pharmaceutical composition comprising both of anitrogen mustard anticancer agent and anti-IL-6 receptor antibody isadministered. In the former two cases, a pharmaceutical compositioncomprising a nitrogen mustard anticancer agent and a pharmaceuticalcomposition comprising anti-IL-6 receptor antibody may be given throughthe same administration route, or a different administration route. Eachof these pharmaceutical compositions is given to cure or inhibit atleast partially the pathological symptoms of patients suffering fromdiseases. The period of administration may be chosen, as appropriate,depending on the age and conditions of the patient.

Preferably, pharmaceutical compositions comprising anti-IL-6 receptorantibody may be administered parenterally, for example via intravenousinjection, drip infusion, intramuscular injection, intraperitonealinjection, subcutaneous injection, and the like, either systemically orlocally. As local dosage forms, preferably, external preparations, localinjections, and the like are used. External preparations are chosen fromliniments such as ointments, gel, cream, emulsions, and liquids, tapes,plaster tapes such as patches, or nebulas such as sprays and powders.

The effective dosage of anti-IL-6 receptor antibody is chosen from therange of 0.001 mg to 1000 mg per kg of body weight per day. Preferably,the dosage is selected from the range of 0.01 to 50 mg per body weight.The above doses depend on the pathological conditions, and hence theyare not limited to these values. The number of administration is usuallyselected from, but not limited to, once or twice per day, once per twoto a few days, or once per one to four weeks.

Pharmaceutical compositions comprising a nitrogen mustard anticanceragent are preferably administered orally, but depending on the nature ofthe active ingredient, the conditions of patients, and the like, theymay be given parenterally as well. For example intravenous injection,drip infusion, intraarterial injection, intramuscular injection,intratumor injection, intrathoracic injection, or intraperitonealinjection, either systemically or locally.

The effective dosage of nitrogen mustard anticancer agents is differenton their kind, but for melphalan, for example, oral administration of 1to 20 mg per day, every day or 1 to 6 times per week, or as high-doseintravenous injection or infusion, single or multiple doses of 20 to 200mg/m² is employed. For cyclophosphamide, oral or intravenousadministration of 50 to 2000 mg per dose usually for one to 5 times perweek, to once per two weeks to one month is employed. The number and theschedule of administration are not limited to those mentioned above.Nitrogen mustard anticancer agents may be given not only alone but alsoin combination with vincristine, adriamycin, prednisolone, and the like,as appropriate.

When a pharmaceutical composition comprising a nitrogen mustardanticancer agent is administered simultaneously with anti-IL-6 receptorantibody, the ratio, is, when combined with daily oral administration ofmelphalan, 0.01 to 1000 fold (weight ratio) relative to the dose ofmelphalan, though it is different on the conditions of the patient andthe administration schedule. Alternatively a pharmaceutical compositioncomprising a certain ratio of the two agents may be administered.However, as mentioned above, the dose ratio varies with the conditionsof the patient etc., and hence it is not limited to the ratio mentionedabove.

It is also possible to set up a schedule in which a pharmaceuticalcomposition comprising a nitrogen mustard anticancer agent and anti-IL-6receptor antibody ate given at different time points. For example, topatients for whom remission was introduced by applying a nitrogenmustard anticancer agent or a combined therapy including the agent as aconstituent element, anti-IL-6 receptor antibody can be administered inorder to maintain remission. Furthermore, administration of a nitrogenmustard anticancer agent or a combined therapy having the agent as aconstituent element and administration of anti-IL-6 receptor antibodymay be repeated every 1 to 4 weeks. For a nitrogen mustard anticanceragent and anti-IL-6 receptor antibody, preferably the former is givenfirst, but the latter may be given first depending on the conditions ofthe patient etc.

Pharmaceutical compositions of the present invention comprising nitrogenmustard anticancer agents, pharmaceutical compositions comprisinganti-IL-6 receptor antibody, and pharmaceutical compositions comprisinga nitrogen mustard anticancer agent and anti-IL-6 receptor antibody ofthe present invention may contain pharmaceutically acceptable carriersand/or additives depending on the route of administration.

Examples of such carriers or pharmaceutical additives include water, apharmaceutically acceptable organic solvent, collagen, polyvinylalcohol, polyvinylpyrrolidone, a carboxyvinyl polymer, sodiumcarboxymethylcellulose, sodium, polyacrylate sodium alginate,water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethyl cellulose, xanthan gum, gum Arabic, casein, gelatin,agar, diglycerin, glycerin, propylene glycol, polyethylene glycol,Vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin(HSA), mannitol, sorbitol, lactose, pharmaceutically acceptablesurfactants and the like.

Actual additives are chosen from, but not limited to, the above orcombinations thereof depending on the dosage form of a therapeutic agentof the present invention.

The present invention also encompasses a simultaneous or sequentialcombined administration of a pharmaceutical agent of the presentinvention with another agent, a biological agent, or a synthetic agent.Other agents are selected from anti-inflammatory agents, antiallergicagents, anti-platelet agents, other anticancer agents or those thatenhance or supplement the activity of the object of the presentinvention.

EXAMPLES

The present invention will now be explained hereinbelow in more detailwith reference to the following reference examples, experimentalexamples and working examples. It is to be noted that the presentinvention is not limited to these examples in any way.

Reference Example 1

Construction of the Anti-IL-6 Receptor Antibody PM-1

The anti-IL-6 receptor antibody MT18 prepared by the method of Hirata etal. (J. Immunol. (1989) 143, 2000-2006) was bound to CNBr-activatedSepharose 4B (manufactured by Pharmacia Fine Chemicals, Piscataway,N.J.) in accordance with the attached directions to purify IL-6 receptor(Yamasaki et al., Science (1988) 241, 825-828).

Thus, a human myeloma cell line U266 was solubilized in 1 mMp-paraaminophenylmethane sulphonylfluoride hydrochloride (manufacturedby Wako Chemicals) (digitonin buffer) containing 1% digitonin(manufactured by Wako Chemicals), 10 mM triethanolamine (pH 7.8), and0.15M NaCl, and then mixed with MT18 antibody conjugated to Sepharose 4Bbeads. The beads were then washed six times with the digitonin buffer toprepare partially purified IL-6 receptor to be used for immunization.

BALB/c mice were immunized four times every 10 days with theabove-mentioned partially purified IL-6 receptor obtained from 3×10⁹U266 cells, and then hybridomas were prepared in a conventional method.A culture supernatant of hybridomas from growth-positive wells wasevaluated for its ability of binding to IL-6 receptor by the methoddescribed below. 5×10⁷ U266 cells were labeled with ³⁵S-methionine (2.5mCi) and were solubilized in the above digitonin buffer.

The solubilized U266 cells were mixed with 0.04 ml of MT18 antibodyconjugated to Sepharose 4B beads and then washed six times in thedigitonin buffer. Using 0.25 ml of the digitonin buffer (pH 3.4),³⁵S-methionine labeled IL-6 receptor was eluted, which was neutralizedwith 0.025 ml of 1M Tris, pH 7.4. The hybridoma culture supernatant 0.05ml was mixed with 0.01 ml Protein G Sepharose (manufactured byPharmacia).

After washing, the Sepharose was incubated with 0.005 ml solution of³⁵S-labeled IL-6 receptor prepared above. Immunoprecipitating substanceswere analyzed by SDS-PAGE to search the culture supernatants ofhybridoma that reacts with IL-6 receptor. As a result, areaction-positive hybridoma clone PM-1 was established. The anti-IL-6receptor antibody PM-1 produced from the hybridoma PM-1 had the IgG1 κsubtype.

The activity of the antibody produced by the hybridoma PM-1 to inhibitthe binding of IL-6 to IL-6 receptor was evaluated using a human myelomacell line U266. Recombinant human IL-6 was prepared from E. coli (Hiranoet al., Immunol. Lett. (1988) 17, 41), and was labeled with ¹²⁵I usingthe Bolton-Hunter reagent (New England Nclear, Boston, Mass.) (Taga etal., J. Exp. Med. (1987) 166, 967). 4×10⁵ U266 cells were cultured witha culture supernatant of 70% (v/v) hybridoma PM-1 and 14000 CPM of¹²⁵I-labeled IL-6 at room temperature for one hour in the presence of a100-fold excess of non-labeled-IL-6. Seventy microliters of a sample waslayered onto 300 μl of FCS in a 400 μl microfuge polyethylene tube,centrifuged, and then the radioactivity of the cells was measured. Theresult revealed that the antibody produced by the hybridoma PM-1inhibits the binding of IL-6 to IL-6 receptor.

Reference Example 2

Construction of a Reshaped Human PM-1 Antibody

A reshaped human PM-1 antibody was obtained by the method described inInternational Patent Application WO 92-19759. From the hybridoma PM-1prepared in Reference example 1, total RNA was prepared in theconventional, method, from which single-stranded cDNA was synthesized.By the polymerase chain reaction (PCR) method, DNA encoding the V regionof mouse PM-1 antibody was amplified. The primers used in the PCR methodare those described in Jones, S. T. et al., Bio/Technology (1991) 9,88-89, 1991.

The PCR-amplified DNA fragments were purified to obtain DNA fragmentscontaining the gene encoding the V region of mouse kappa-type L chainand DNA fragments containing the gene encoding the V region of mousegamma-type H chain. These DNA fragments were ligated to a plasmid pUC19,which was then transfected into competent E. coli cells CH5α to obtainan E. coli transformant. From the transformant thus obtained, the aboveplasmid was obtained, and the base sequence of the V region-codingregion in the plasmid was determined in a conventional method, and thecomplementarity determining region (CDR) of each V region wasidentified.

In order to construct vectors that express chimera PM-1 antibody, cDNAsencoding the V region of κ L chain and H chain of mouse PM-1 wereseparately inserted into HCMV expression vectors. In order to constructa reshaped human PM-1 antibody, the CDR of V region of mouse PM-1 wasimplanted to human antibody by the CDR grafting method. In order for theCDR of human antibody to form appropriate antigen-binding sites,substitution of amino acids of the framework region (FR) of antibody Vregion was conducted.

In order to express genes of L chain and H chain of the reshaped humanPM-1 antibody thus constructed, DNA encoding the L chain or the H chainwas separately inserted into a vector containing the human elongationfactor 1α (HEF-1α) promoter, and a vector expressing the L chain or theH chain of the reshaped human PM-1 (hPM-1) antibody was constructed. Bysimultaneously inserting these two expression vectors into CHO cells, acell line that produces reshaped human PM-1 antibody (hPM-1) wasestablished. The ability of hPM-1 thus obtained to bind to human IL-6receptor was confirmed by ELISA. Furthermore, hPM-1 inhibited thebinding of human IL-6 to human IL-6 receptor in a similar manner to thatof mouse antibody and chimeric antibody.

Example 1

The Effects of Combined Use of Anti-human IL-6 Receptor Antibody and aChemotherapeutic Agent on the Growth of Human Myeloma Cells

The effects of anti-human IL-6 receptor antibody on sensitivity of KPMM2cells to chemotherapeutic agents used for treatment of myeloma such asadriamycin (ADR, manufactured by Kyowa Hakko), vincristine (VCR,manufactured by Sigma Chemical Co.), and melphalan (L-PAM, manufacturedby Sigma Chemical Co.) were evaluated.

KPMM2 is a multiple myeloma cell line derived from the ascites of ahuman patient with myeloma (see Japanese Unexamined Patent Publication(Kokai) No. 7-236475). The patient with myeloma had maintained remissionby the MCNU (ranimustine) and MP (melphalan, prednisolone) therapy, butthe disease recurred and the subsequent VAD (vincristine, adriamycin,dexamethasone) therapy was ineffective. The growth of KPMM2 cells ispromoted by IL-6 and is markedly inhibited by anti-IL-6 antibody oranti-IL-6 receptor antibody (Rinsho Ketueki [The Japanese Journal ofClinical Hematology] (1994) 35, 1361-1365). The growth activity of thecell was evaluated by incorporation of ³H-labeled thymidine(manufactured by Amersham) into the cell.

The KPMM2 cells that had been maintained were washed thoroughly with afresh medium (RPMI1640 medium supplemented with 20% FBS), and then wereadjusted to 4×10⁵/ml, which was dispensed in 50 μl aliquots in a 96-wellflat-bottomed microtiter plate (manufactured by Falcon). Furthermore, amedium containing a recombinant human IL-6 (Asagoe, Y. et al.,Bio/Technology (1988) 6, 806-809), anti-human IL-6 receptor antibodyhPM-1 (see the above reference example 1 or International PatentApplication WO 92-15759), and the above chemotherapeutic agents or afresh medium as control were added to make 200 μl in each well.

The plate was incubated at 37° C. for 4 days under the humidifiedcondition in the presence of 5% CO₂. At four hours before the end ofincubation, 10 μl of ³H-labeled thymidine solution (100 μCi/ml) wasadded to each well, and then incubated for more 4 hours. At the end ofincubation, the cells were collected onto the glass filter (manufacturedby Printed Filtermat A, WALLAC) using a harvester (Micro 96 Harvester,manufactured by SKATRON Instruments). The radioactivity incorporatedinto the cells was measured by a microbeta (1450 MicroBeta, manufacturedby WALLAC).

The activity of growth inhibition on KPMM2 was expressed using theeffect by a chemotherapeutic agent alone as the control. Using theincorporation of ³H-labeled thymidine into the cell for eachexperimental group in which a each concentration of a chemotherapeuticagent was added as 100, the amount of ³H-labeled thymidine incorporatedinto the cell in an experimental group in which anti-human IL-6 receptorantibody was simultaneously added was compared as an index.

As a result, comparison of the relationship with the concentration of achemotherapeutic agent in the presence of a fixed concentration ofanti-human IL-6 receptor antibody revealed that the index of adriamycinand vincristine was almost constant regardless of their concentration(FIG. 3 to FIG. 6), whereas the index decreased with the increase inmelphalan concentration (FIG. 1 and FIG. 2). In the presence of 1 ng/mlIL-6, the growth index of 10 μg/ml anti-IL-6 receptor antibody alone was33.9, whereas it dropped to 15.5 in the co-existence of 1 μg/mlmelphalan. Similar results were obtained in the presence of 0.1 μg/ml ofIL-6 and the index of the antibody alone was 28.4, whereas in theco-existence of 1 μg/ml melphalan, it was 15.9. Thus, combined use ofanti-IL-6 receptor antibody and melphalan was shown to have asynergistic effect.

Example 2

The Effects of Combined Use of Anti-human IL-6 Receptor Antibody and aChemotherapeutic Agent in a Human Myeloma Cell Implanted SCID MouseSystem

It was shown in working example 1 that anti-human IL-6 receptor antibodyenhances the antitumor effect of chemotherapeutic agents. Among them,melphalan (manufactured by Sigma Chemical Co.) which was found to act ina synergistic manner was used to study an in vivo effect of combineduse.

For evaluation of antitumor activity, a xenograft model was used. Thus,KPMM2, a human myeloma cell line derived from ascites of a patient withmultiple myeloma was implanted to a male SCID mouse (FOX CHASE C.B17/Icr-Scid Jcl, purchased from Nippon Klea) via the tail vein. At thistime, the tumor cells grow in the bone marrow and come to producemyeloma protein (M protein) in the peripheral blood. Furthermore, thismodel system is very close to an actual clinical condition in that itdevelops major symptoms of multiple myeloma in humans such as bonedisorders, elevated blood calcium, and the like.

Single cell suspension of myeloma cells for implantation was prepared bypassing through a mesh the well minced KPMM2 cells that have beenmaintained in vivo. Cell density was adjusted to 3×10⁷/ml, which wasimplanted at 0.2 ml per mouse via the tail vein (6×10⁷ cells per mouse).The day when the cells were implanted was set at day 0.

With regard to anti-human IL-6 receptor antibody, a stock solution thathad been preserved at 12.1 mg/ml was diluted in sterile phosphate bufferto make 5 mg/ml. This was given to mice at 0.2 ml per animal via thetail vein on day 8 (1 mg per mouse). The control group received sterilephosphate buffer containing no antibody in a similar manner.

Melphalan (L-PAM, manufactured by Sigma Chemical Co.) that was suspendedat 0.3 or 0.1 mg/ml in 0.2% CMC (carboxymethylcellulose) solution inwater was used. This was given orally at 0.1 ml per 10 g body weight ofmouse (3 or 1 mg/kg weight) for 5 consecutive days starting on day 1.The control group received 0.2% CMC solution in water containing nomelphalan in a similar manner.

The experiment was carried out on the following six groups: (1) themelphalan and antibody non-administration group; (2) the 1 mg/kgmelphalan single-drug administration group; (3) the 3 mg/kg melphalansingle-drug administration group; (4) antibody single-drugadministration group; (5) the 1 mg/kg melphalan and antibody combinedadministration group; and (6) the 3 mg/kg melphalan and antibodycombined administration group. Group 1 included 9 mice, whereas each ofthe other groups included 7 mice. Furthermore, tumor was not implantedon mice of the same lineage and purchased on the same day, which wereused as negative control for M protein detection.

Indices of the drug efficacy included the survival period, the survivalratio without disease on day 120, and the amount of M protein on day 30.For analysis using a survival curve, a generalized Wilcoxon's test (SPSSfor windows ver. 6, SPSS inc.) was used. A significance level of 5% orlower was considered to be significant.

Serum M protein was detected as human IgG using the ELISA method. First,mouse serum was dispensed into a 96-well micro plate that had previouslybeen coated with anti-human IgG antiserum and was allowed to stand. Thenalkaline phosphatase-conjugated human IgG antibody was bound thereto,and SIGMA104-phosphatase substrate was added for color development,absorbance of which was read using a microplate reader. M proteincontent in the serum was calculated using the standard curve obtainedfrom normal human IgG.

In the anti-IL-6 receptor antibody single-drug administration group orthe 1 mg/ml melphalan administration group, no life elongation effectwas observed as compared to the non-administration group. But thecombined use of them significantly elongated life span as compared tothe non-administration group or both of the single-drug administrationgroups (FIG. 7). Furthermore, in the measurement of M protein in theserum on day 30 after implantation, the combined use of them reduced Mprotein level (FIG. 8). In the case of 3 mg/kg melphalan, a significantlife elongation effect was observed for the melphalan single-drugadministration group as compared to the non-administration group, but byusing antibody in combination a significant life elongation effect waseven observed compared to the single-drug administration (FIG. 9). Thesurvival ratio without disease on day 120 was 2/7 in the 3 mg/kgmelphalan single-drug administration group, but the ratio improved to4/7 in the antibody combined administration group (Table 1).

The administration of melphalan caused toxicity in mice and inhibitedbody weight gain (FIG. 10). When melphalan was used in combination withanti-IL-6 receptor antibody, an antitumor effect was enhanced but it didnot expand toxicity (inhibition of body weight gain).

TABLE 1 Extension of survival period by combined use of anti-human IL-6receptor antibody hPM-1 and melphalan hPM-1 Life- None adminis-Melphalan Survival elongation disease tration dosage n period ratio* (%)ratio — CMC control 9 46.9 ± 1.5 100 0/9 — 1 mg/kg 7 47.3 ± 3.1 101 0/7— 3 mg/kg 7 84.1 ± 9.1 175 2/7 day 8 CMC control 7 55.1 ± 5.2 118 0/7day 8 1 mg/kg 7 68.6 ± 7.5 146 0/7 day 8 3 mg/kg 7 110.0 ± 5.1  235 4/7Life elongation ratio*: 100 × (drug administration group/drugnon-administration group). Survival period was expressed as mean ±standard error.

It was revealed that by using anti-IL-6 receptor antibody and melphalanin combination, the life elongation effect was significantly enhanced inKPMM2 which is a cell line derived from a patient who was resistant toMP and VAD therapy.

Example 3

The Effects of Combined Use of Anti-human IL-6 Receptor Antibody and aChemotherapeutic Agent in a Human Myeloma Cell-implanted SCID MouseSystem—a Study on in vivo Dose Dependency of Antibody

Example 2 has shown that anti-human IL-6 receptor antibody enhances theantitumor effect of melphalan. Accordingly, we studied dose dependencyof anti-human IL-6 receptor antibody in the combined administration ofanti-human IL-6 receptor antibody and melphalan.

For evaluation of antitumor effects, a xenograft model animal that wasproduced by implanting KPMM2 cells via the tail vein was used as inWorking example 2. Thus, single-cell suspensions which were prepared bypassing through a mesh KPMM2 cells that were minced after having beenmaintained in vivo were implanted at 0.2 ml per mouse via the tail vein(6×10⁶ cells per mouse). The day when the cells were implanted was setat day 0.

With regard to anti-human IL-6 receptor antibody hPM-1, a stock solutionthat had been preserved at 6.57 mg/ml was diluted in sodium phosphatebuffer to make each solution of 5, 1, 0.2, and 0.04 mg/ml. They weregiven to mice at 0.1 ml per 10 g body weight of mouse via the tail veinon day 14 to create the 50, 10, 2, and 0.4 mg/kg administration groups.The control group received the same buffer without antibody in a similarmanner.

Melphalan (L-PAM, manufactured by Sigma Chemical Co.) was used as asuspension of 0.1 mg/ml in 0.2% CMC solution in water. This was givenorally at 0.1 ml per 10 g body weight of mouse (1 mg/kg weight) for 5consecutive days starting on day 7. The control group received 0.2% CMCsolution in water without melphalan in a similar manner.

The experiment was carried out on the following 10 groups: (A) themelphalan and the antibody non-administration group; (B) the melphalansingle-drug administration group; (C) the each dose of antibodysingle-drug administration group, 4 groups (50, 10, 2, 0.4 mg/kgweight); and, (D) the melphalan and the each dose of antibody combinedadministration group, 4 groups (50, 10, 2, 0.4 mg/kg weight). Thenon-administration group included 12 mice per group, the melphalanadministration group 6 mice per group, whereas each of the other groupsincluded 7 mice. Furthermore, mice of the same lineage and purchased onthe same day were raised without implanting a tumor, and the animalswere used as negative control for M protein detection. The amount of Mprotein was calculated as described in Example 2.

Indices of the drug efficacy included the survival period, and theamount of M proteins on day 30, day 35, and day 42. For analysis of thesurvival period, a generalized Wilcoxon's test (SPSS for Windows ver. 6,SPSS inc.) was used. A significance level of 5% or lower was consideredto be significant. For analysis of the amount of M protein in the serum,the ANOVA (Analysis of variance, SPSS for windows ver. 6, SPSS inc.) wasfirst conducted. After confirming significance, Bonferroni method (SPSSfor windows ver. 6, SPSS inc.) was used, and a significance level of 5%or lower was considered to be significant.

Since death cases occurred on day 35 in the non-administration group andthe each dose of antibody single-drug administration group, they werecompared for the amount of M-protein on day 30. On the other hand, inthe melphalan single-drug administration group, and the melphalan andantibody combined administration group, the amount of M protein on day30 was very low. Since the anti-human IL-6 receptor antibody that hadbeen administered was detected as M protein thus affecting the assay,they were compared for the data on day 35 and day 42.

On day 30 in the antibody single-drug administration group, none of thedoses significantly inhibited the amount of M protein, but thesingle-drug administration of melphalan significantly inhibited this(FIG. 11). Then, on day 35 and 42, the effect of combined use ofantibody administration relative to the melphalan administration wasstudied. The result revealed that the combined use of anti-human, IL-6receptor antibody at 10 mg/kg, 2 mg/kg, and 0.4 mg/kg significantlyreduced the amount of M protein (FIGS. 12, 13).

For the survival period, none of the doses in the antibody single-drugadministration group gave a significant life elongation effect.Furthermore, melphalan alone has shown a significant life elongationeffect, which was further enhanced by combined use of antibody (Table2). At any dose, the life elongation effect tended to increase.Furthermore, in the 0.4 mg/kg and 50 mg/kg administration groups,significance was observed relative to the melphalan single-drugadministration group in the generalized. Wilcoxon's test (FIG. 14).

TABLE 2 Extension of survival period by combined use of anti-human IL-6receptor antibody hPM-1 and melphalan hPM-1 Life- adminis- MelphalanSurvival elongation tration dosage n period ratio (%) — CMC control 1241.0 ± 1.7 100 50 mg/kg CMC control 7 41.6 ± 1.0 101 10 mg/kg CMCcontrol 7 40.4 ± 1.7  99  2 mg/kg CMC control 7 41.4 ± 1.3 101 0.4mg/kg  CMC control 7 40.3 ± 1.2  98 — 1 mg/kg 6 58.2 ± 1.8 142 (100) 50mg/kg 1 mg/kg 7 65.7 ± 3.9 160 (113) 10 mg/kg 1 mg/kg 7 64.3 ± 2.7 157(111)  2 mg/kg 1 mg/kg 7 63.3 ± 2.3 154 (109) 0.4 mg/kg  1 mg/kg 7 63.7± 1.1 155 (110) Life elongation ratio: 100 × (drug administrationgroup/drug non-administration group). Values in the parentheses wereobtained using the melphalan single-drug administration group ascontrol. Survival period was expressed as mean ± standard error.

From the foregoing, it was demonstrated, the combined use of anti-IL-6receptor antibody and melphalan has shown an antitumor effect at anydose from 0.4 mg/kg through 50 mg/kg.

The administration of melphalan caused toxicity to mice and inhibitedbody weight gain. When melphalan was used in combination with anti-humanIL-6 receptor antibody, an antitumor effect was enhanced but toxicity(inhibition of body weight gain) was not expanded. Therefore, it wassuggested that in the treatment of myeloma the administration ofmelphalan may be useful in enhancing effects, reducing the dosage, andbreaking melphalan-resistance. Reference to microorganisms depositedunder Patent Cooperation Treaty Rule 13(2): The name and address of thedepository institute Depository organization: the National Institute ofBioscience and Human Technology,

Agency of Industrial Science and Technology Address: 1-3, Higashi1-chome, Tsukuba-shi, Ibaraki, Japan

Accession No. Date deposited FERM BP-2998 July 10, 1990

Depository organization: National Collections of Industrial and MarineBacteria Limited Address: 23 St Macher Drive, Aberdeen AB2 IRY, UNITEDKINGDOM

NCIMB 40366 Feb. 11, 1991 NCIMB 40362 Feb. 11, 1991

What is claimed is:
 1. A method for treatment of myeloma which compriseadministering a reshaped human PM-1 antibody with melphalan to a subjectin need of such treatment.
 2. The method of 1 wherein the reshaped humanPM-1 antibody is the antibody hPM-1.